Method for mammalian cell separation from a mixture of cell populations

ABSTRACT

The invention provides a method for both the positive and negative selection of at least one mammalian cell population from a mixture of cell populations utilizing a magnetically stabilized fluidized bed. One desirable application of this method is the separation and purification of hematopoietic cells. Target cell populations include human stem cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the national phase application filed under 35 U.S.C.§371(b) of international application No. PCT/US94/11104, filed Sep. 6,1994, which in turn is a continuation-in-part of U.S. Ser. No.08/130,094, filed Sep. 30, 1993, now U.S. Pat. No. 5,409,813, issuedApr. 25, 1995.

TECHNICAL FIELD

This invention relates to the use of a magnetically stabilized fluidizedbed (MSFB) for selective separation and purification of mammalian cellpopulations.

BACKGROUND ART

Separation of mixtures of chemicals, biomolecules and cell types isoften effected by immunoaffinity chromatography. Packed beds, such asthose used in column chromatography, are often used in affinityseparation. However, problems such as nonspecific trapping or filtrationof cells and clogging make the use of a packed bed undesirable for cellseparation. In addition, when fine particles are used to increase themass transfer efficiency of packed beds, a large pressure drop acrossthe bed often results. These problems require significant washing of thepacked bed in order to flush contaminants and other cellular debris fromthe column.

One device that has been developed for reducing the pressure drop acrossa column of particles is the fluidized bed. A fluidized bed consists ofsolid particles and a gas or liquid which is passed upwardly through theparticle bed with velocity sufficient to ensure that the drag forces ofthe fluid counterbalance the gravitational forces on the particle andcause random motion of the particles. The bed of particles will becomefluidized and expand, resulting in a lower pressure drop across thefluidized bed as compared to the pressure drop across a packed bed ofthe same height. The fluidization of the bed also provides more surfacecontact between the particle and the fluid passing through the bed.

One disadvantage associated with fluidized beds is the radial and axialmovement of the particles which result in significant intermixing of theparticles. An advancement in fluidized bed technology is themagnetically stabilized fluidized bed (MSFB) which involves the use of amagnetic field and magnetizable particles to stabilize the bed. It hasbeen found that, by supplying a magnetic field parallel to the path offluid flow, the magnetizable particles can be locked in place, thuseliminating the intermixing of the particles.

In general, the MSFB combines some of the best characteristics of thefluidized bed with those of a fixed bed. More particularly, the MSFBprovides a low pressure drop, the ability to transport solids through asystem and good mass transfer driving force even as the fluid isdepleted of its source. See Burns, Structural Studies of aLiquid-Fluidized Magnetically Stabilized Bed, Chem. Eng. Comm.,67:315-330 (1988). All documents cited herein are hereby incorporated byreference.

Because of these advantages, MSFBs have been used to separate variouschemical species and proteins, and to filter yeast. For example, for theuse of a MSFB to separate proteins see Burns, et al., Application ofMagnetically Stabilized Fluidized Beds to Bioseparations, ReactivePolymers, 6:45-50 (1987) (human serum albumin); Lochmuller et al.,Affinity Separations in Magnetically Stabilized Fluidized Beds:Synthesis and Performance of Packing Materials, Separation Science andTechnology; 22:2111-2125 (1987) (trypsin); and U.S. Pat. No. 5,130,027,issued to Noble, Jul. 14, 1992, (Cytochrome-C). The use of a MSFB toseparate various organic and inorganic compounds is discussed in U.S.Pat. No. 5,084,184 issued to Burns on Jan. 28, 1992. Finally, the use ofan MSFB as a filter to collect yeast cells was reported by Terranova etal. Continuous Cell Suspension Processing using Magnetically StabilizedFluidized Beds, Biotechnology and Bioengineering, 37:110-120 (1991). Inthis latter reference, the filtration was not based on immunoaffinitybut rather on electrostatic interaction between the positively chargednickel particles contained in the MSFB and the negatively charged yeastcells.

However, none of these references discuss the use of the MSFB for theaffinity separation of mammalian cell population from a mixture of cellpopulations. The separation of a particular mammalian cell populationfrom a mixture of cell populations is quite different from theseparation of chemical species such as proteins from a solution. Mostmammalian cells are on the order of 8 to 20 microns (μ) in diameter. Incontrast, the proteins and other chemical species which have beenseparated in a MSFB to date are significantly smaller, i.e. on the orderof 1000 fold or more. Thus, the probability that the larger mammaliancell with greater fluid drag will bind to the particle and be retainedis significantly lower under similar conditions. In addition, anotherfactor unique to the separation of mammalian cells is the need topreserve cell viability.

In contrast to yeast cells, which are relatively insensitive to changesin osmolarity, pH and shear, higher order mammalian cells are much moresensitive to shear forces exerted during purification, pH osmolarity,and the chemical composition of the reagents used. Therefore, both thesteps comprising the method and all reagents used must be non-toxic tothe cells.

Mammalian hematopoietic (blood) cells provide a diverse range ofphysiological activities. Blood cells are divided into lymphoid, myeloidand erythroid lineages. The lymphoid lineages, comprising B cells and Tcells, provides for the production of antibodies, regulation of thecellular immune system, detection of foreign agents in the blood,detection of cells foreign to the host, and the like. The myeloidlineage, which includes monocytes, granulocytes, megakaryocytes as wellas other cells, monitors for the presence of foreign bodies, providesprotection against neoplastic cells, scavenges foreign materials,produces platelets, and the like. The erythroid lineage provides the redblood cells, which act as oxygen carriers.

Despite the diversity of the nature, morphology, characteristics andfunction of the blood cells, it is presently believed that these cellsare derived from a single progenitor population, termed "stem cells."Stem cells are capable of self-regeneration and may become lineagecommitted progenitors which are dedicated to differentiation andexpansion into a specific lineage.

A highly purified population of stem cells is necessary for a variety ofin vitro experiments and in vivo indications. For instance, a purifiedpopulation of stem cells will allow for identification of growth factorsassociated with their self-regeneration. In addition, there may be asyet undiscovered growth factors associated (1) with the early steps ofdedication of the stem cell to a particular lineage; (2) the preventionof such dedication; and (3) the negative control of stem cellproliferation.

Stem cells find use: (1) in regenerating the hematopoietic system of ahost deficient in stem cells; (2) in a host that is diseased and can betreated by removal of bone marrow, isolation of stem cells and treatmentof individuals with drugs or irradiation prior to re-engraftment of stemcells; (3) producing various hematopoietic cells; (4) detecting andevaluating growth factors relevant to stem cell self-regeneration; (5)the development of hematopoietic cell lineages and assaying for factorsassociated with hematopoietic development; and, is a target for genetherapy to endow blood cells with useful properties.

Highly purified stem cells are essential for hematopoietic engraftmentincluding but not limited to that in cancer patients and transplantationof other organs in association with hematopoietic engraftment. Stemcells are important targets for gene therapy, where the inserted genespromote the health of the individual into whom the stem cells aretransplanted. In addition, the ability to isolate the stem cell mayserve in the treatment of lymphomas and leukemias, as well as otherneoplastic conditions. Thus, there have been world-wide efforts towardisolating the human hematopoietic stem cell in substantially pure orpure form.

Stem cells constitute only a small percentage of the total number ofhematopoietic cells. Hematopoietic cells are identifiable by thepresence of a variety of cell surface "markers." Such markers may beeither specific to a particular lineage or progenitor cell or be presenton more than one cell type. Currently, it is not known how many of themarkers associated with differentiated cells are also present on stemcells. One marker which was previously indicated as present solely onstem cells, CD34, is also found on a significant number of lineagecommitted progenitors. U.S. Pat. No. 4,714,680 describes a compositioncomprising human stem cells.

Hematopoietic cells are initially obtained as a mixture of a variety ofcell populations ("mixture of cell populations"). The cell population tobe purified or enriched for is termed herein the "target" cellpopulation. Separation techniques involve successive purification stepsrelying on the use of affinity matrices to either retain nontarget cellsand allow the target cells to flow through (negative selection) or toretain the target cells and allow the nontarget cells to flow through(positive selection).

Typically, hematopoietic cells are separated using negative selectionaffinity separations which are performed in a batch mode. For example,in processing a normal bone marrow (BM) harvest, it may be desirable toobtain only those cells exhibiting a specific cell surface antigen suchas the CD34 antigen (CD34⁺ cells) which includes the stem cellpopulation and a variety of other, more differentiated, cells.Typically, only approximately 0.1 to 5% of the total mononuclear cellpopulation in a blood sample express the CD34 antigen. In contrast,cells expressing the CD15⁺ cell surface antigen, i.e. CD15 cells, whichare more differentiated than stem cells, comprise approximately 50 to75% of the total mononuclear cells.

In order to separate the target CD34⁺ cells from the mixture of cellpopulations by negative selection, the cell sample is placed in a vesselcontaining beads conjugated to an antibody specific to CD15. CD15⁺ cellsbind to the anti-CD15 antibody and are then removed from solution byremoval of the beads. Thus, by depleting the CD15⁺ cells, less than halfto one quarter of the original cells, including the target CD34⁺ cells,remain for additional processing. Negative selection has been essentialin separating stem and/or progenitor cells from BM or other sourcessince the target cells are present in such a low concentration.

In contrast, positive selection refers to a process in which the targetcell population is bound to a particle having affinity for the targetcell population and the nontarget cell populations do not bind and flowthrough. The target cell population is then obtained by releasing thecells from the particles and collecting the cells.

Because the target cell population typically comprises only a smallfraction of the mixture of cell populations, positive selection would bethe preferred process if it could be made efficient enough toselectively remove such a small percentage of cells. As noted earlier,CD34⁺ cells comprise only approximately 0.1 to 5% of the totalmononuclear cell population. Of these cells stem cells comprise only asmall percentage. Considering the time and reagents needed to stain andsort cells, it would be advantageous to directly select CD34⁺ cells forsubsequent staining and sorting instead successive purification of themixture of cell populations that typically result from negativeselection methods.

Until recently, positive selection of human hematopoietic cells has notbeen possible. One method which has been developed is the Ceprate LC®system which uses a packed bed column and an avidin-biotin affinitysystem to select CD34⁺ cells. In this system, the avidin protein isattached to a bead or other solid support. The suspension containing thetarget CD34⁺ cells is mixed with a biotin-conjugated anti-CD34-antibodyunder conditions which allow the antibody to bind to CD34⁺ cells. Thissuspension is then passed downwardly through the column and, due to theaffinity of the biotin to avidin, the CD34⁺ cells adhere to the supportbeads. After the entire suspension has passed through the column, thecolumn is washed to remove any excess suspension or impurities. Thesupport beads with the attached CD34⁺ cells are then agitated tophysically separate the CD34⁺ cells from the beads. Although this methodallows for positive selection of CD34⁺ cells, it also results insignificant non-specific separation and therefore retention of nontargetcells, such as cancerous cells. It is believed that at least a portionof this contamination occurs due to filtration in the packed column.

Accordingly, it is an object of this invention to provide a method forboth the positive and negative selection of mammalian hematopoieticcells that results in significantly less non-specific cell separationand filtration.

DISCLOSURE OF THE INVENTION

A method is provided for the selective enrichment of at least onemammalian target cell population from a suspension of a mixture of cellpopulations containing the target cell population and at least onenontarget cell population. The method comprises the steps of fluidizinga column containing a bed of magnetizable particles with a firstsolution, said particles comprising a substance having affinity for aspecific population of mammalian cells; stabilizing the fluidized bed ofmagnetizable particles with a uniform magnetic field; and passing asuspension containing the mixture of cell populations through thefluidized, magnetically stabilized bed at a velocity at which at leastone population of cells binds to said particles so as to be retained,thereby enriching the target cell population.

In one embodiment, a positive selection method is provided wherein theparticles have affinity for the target cell population and the methodadditionally comprises the step of collecting the target cell populationwhich are bound to the particles.

In another embodiment, a negative selection method is provided whereinthe particles have affinity for the nontarget cell populations and, themethod additionally-comprises the step of collecting the target cellswhich pass through the fluidized, magnetically stabilized bed.

Another embodiment provides a method for positively separating at leastone target mammalian cell population from a suspension of a mixture ofcell populations containing the target cell population and at least onenontarget cell population. The method comprises the steps of fluidizinga column containing a bed of magnetizable particles with a solution,said particles comprising at least one antibody having affinity for thetarget cell population; stabilizing the fluidized bed of magnetizableparticles with a radially and axially substantially uniform magneticfield; passing a suspension containing the mixture of cell populationsthrough the fluidized, magnetically stabilized bed at a velocity wherebythe particles selectively bind the target cell population; andcollecting the target cell population.

Another embodiment provides a method for negatively separating at leastone mammalian cell population from a suspension of a mixture of cellpopulations containing the target cell population and at least onenontarget cell population. The method comprises the steps of fluidizinga column containing a bed of magnetizable particles with a solution,said particles comprising at least one antibody having affinity fornontarget cells, stabilizing the bed of magnetizable particles with aradially and axially substantially uniform magnetic field, passing asuspension containing the mixture of cell populations through thefluidized, magnetically stabilized bed at a velocity whereby theparticles bind the nontarget cell population, and collecting the targetcell population which pass through the bed.

In yet another embodiment, a method for the selective enrichment of atleast one mammalian target cell population from a suspension of amixture of cell populations is provided, wherein the suspension containsthe target cell population and at least one nontarget cell population.In this embodiment, the method comprises the steps of attaching aplurality of paramagnetic, or preferably superparamagnetic, magnetizablenanoparticles having affinity for at least one specific population ofmammalian cells to these specific populations of cells, fluidizing acolumn containing a bed of magnetizable particles with a first solution,stabilizing the fluidized bed of magnetizable particles with a magneticfield, and passing the suspension through the fluidized, magneticallystabilized bed at a velocity at which at least one specific populationof cells binds to the magnetizable particles through magnetic attractionbetween the magnetizable nanoparticles, and magnetizable particles,thereby enriching the target cell population.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a MSFB apparatus.

FIG. 2 shows a horizontally rotated MSFB for batch cell collection priorto washing and cell removal.

BEST MODE FOR CARRYING OUT THE INVENTION

This invention is directed to a method for selectively separating atleast one specific mammalian cell population from a mixture of cellpopulations. In this method, a protein, antibody, or other chemicalsubstance having a specific affinity for at least one population of themixture of cell populations is attached in any manner known in the artto magnetizable particles under conditions sufficient to allow bindingof the specific cell population. The magnetizable particles form the bedof a MSFB and a suspension containing the mixture of cell populations ispassed through the bed to bind the selected cell population to theparticles. Alternatively a plurality of very small, magnetizable,nanoparticles can be attached to at least one cell population in themixed cell population prior to passing the mixed population of cellsthrough the MSFB. The nanoparticles attach to the cells through abinding moiety that has affinity for at least one of the cellpopulations found in the mixed population. Once these cell/nanoparticlecomplexes are formed, the entire mixed population is passed through theMSFB. In this embodiment, a population of cells can be separated basedon the magnetic attraction between the cell/nanoparticle complex and themagnetizable particles while the magnetic field is activated. When themagnetic field is turned off, the magnetizable particles forming the bedof the MSFB and the nanoparticles attached to the cells lose theirmagnetism and, therefore, become unbound from one another.

As an aid in understanding this invention, the following definitions areprovided. As used herein, the term "target cell population" denotesthose cells which are desirably being purified or enriched. The term"positive selection" refers to a process in which the target cellpopulation is purified or enriched by removing the target cellpopulation from a mixture of cell populations by directly binding thetarget cell population to particles having affinity therefor. Incontrast, the term "negative selection" refers to a process in which thetarget cell population is purified or enriched by removing nontargetcell populations from the mixture of cells by binding the nontarget cellpopulations to particles having affinity therefor.

This invention can be used to separate any type of mammalian cells.Preferably the cells are hematopoietic cells. More preferably they arehuman hematopoietic cells. Possible sources of hematopoietic cellsinclude but are not limited to, bone marrow (BM), both adult and fetal;peripheral blood (PB), or fractions thereof; umbilical cord blood; andcadaveric bone marrow. In general, the cells may be derived from anymammalian organ including, but not limited to, the organs found in thegastrointestinal, circulatory, skeletal, muscular, nervous, skin,respiratory, and reproductive systems. Preferred organs include, but arenot limited to, adult or fetal liver, kidney, and pancreas. A morepreferred source of hematopoietic cells are bone marrow and peripheralblood.

A preferred target cell population is the human stem cell, as definedfor instance in U.S. Pat. No. 5,061,620, the entirety of which is herebyincorporated by reference. Alternatively, a preferred target cellpopulation is any hematopoietic stem cell or any cells that possesslymphoid, myeloid or erythroid characteristics. Another desirable targetcell population is a hematopoietic progenitor cell population.

The affinity binding chemistry used in one embodiment of this inventioncan be between any of the generally known binding pairs including, butnot limited to, antibodies and antigens; hormones and receptors; enzymesand either substrates, coenzymes, inhibitor activators; DNA and itscomplement (a repressor or catabolite gene activator protein for doublestranded DNA or the complement of a single strand of DNA); and messengerRNA and ribosomes.

Preferably the affinity binding pair is specific for mammalian cells.More preferably, an antibody specific for CD34⁺ cells is used. Any ofthe methods known in the art for conjugating an antibody to a solidphase support, such as the magnetizable particle described herein, canbe used in this invention. The amount of antibody will depend on theantibody affinity as well as antigen density on the target cellpopulation. Typically, the maximum number per surface area will be used,which is on the order of about 1.2×10⁶ antibodies/μm². Alternatively, asfew as 1×10³ antibodies/μm² can be used with a suitable antibody.

The binding of the mammalian cells to the magnetizable particles canalso be through one or more binding moieties which are attached to theparticles. For example, a linker may be attached to the particles whichwill specifically bind to an antibody that is specific to a specificmammalian cell.

An alternative embodiment of this invention contemplates the use ofmagnetic attraction to separate the target cells from the mixedpopulation. In this embodiment, a plurality of very small, secondmagnetizable particles are added to the mixed population of cells priorto introducing the mixed population to the MSFB. An example of such verysmall, magnetizable particles are described in U.S. Pat. No. 4,770,183,herein incorporated by reference. A binding moiety that is specific forat least one population of cells is attached to these very smallmagnetizable particles through any of the methods known in the art.Therefore, these very small, magnetizable particles will bind only tocertain populations of cells in the mixed population. For purposes ofclarity, these second magnetizable particles are referred to as"nanoparticles" throughout this application in order to distinguish themfrom the magnetizable particles used in the MSFB. In addition to theirdifferent uses, the nanoparticles are also significantly smaller thanthe magnetizable particles; typically the nanoparticles range in sizefrom 50Å to 5000Å (0.005 to 0.05 microns) as compared to themagnetizable particles in the MSFB, which typically range in size from 5to 500 microns, as discussed in depth below. In addition tonanoparticles, microparticles ranging in size from about 0.5 microns toabout 10 microns could also be attached to the cells and used in thisembodiment of the invention.

In general, the magnetizable nanoparticle is comprised of a core ofparamagnetic or superparamagnetic material surrounded by a coating.Preferably a superparamagnetic material is used. The coating is madefrom a biologically compatible support material such as any stablepolymer, carbohydrate, or protein. A monoclonal antibody (mAb) specificfor at least one target cell population, for example a CD34 mAb, isattached to this coating through any of the methods known in the art.Because this mAb is specific for only certain cell population(s), alarge number of the nanoparticles will attach to only these cellpopulations. Saturation of all available antibody binding sites may ormay not be required. When these cell/nanoparticle complexes are passedthrough the MSFB, the magnetic attraction between the cell/nanoparticlecomplex and the magnetizable particles will hold the cell/nanoparticlecomplex next to the magnetizable particles. The cell/nanoparticlecomplex can then be released from the magnetizable particles by simplyremoving the magnetic field, if certain paramagnetic orsuperparamagnetic particles are used.

Removal of the nanoparticles from the cells may be unnecessary becauseof their extremely small mass and/or the cells' ability and propensityto expel the nanoparticles after a certain period of time.Alternatively, a biodegradable material could be used in the outercoating of the nanoparticle, thus allowing the nanoparticles to bereleased from the cells once this layer degrades.

It is contemplated that this inventive method may be used for eitherpositive or negative selection of any mammalian cells. One preferred useof this invention is for the positive selection of human hematopoieticcells. The selected hematopoietic cells could then be further purifiedusing any suitable technique including, but not limited to, thatdescribed in "A Method for Producing a Highly Enriched Population ofHematopoietic Stem Cells and Stem Cell Populations derived Therefrom,"as described in co-pending U.S. patent application Ser. No. 08/112,603(now abandoned), or placed in culture and expanded in number accordingto any suitable method including, but not limited to, co-pending U.S.patent application Ser. Number 07/846,782 (now abandoned), entitled"Culturing of Hematopoietic Stem Cells and Their Genetic Engineering."The entirety of both these co-pending applications is herebyincorporated by reference.

Although positive selection is a desired mode of operation, other modesof operation are also envisioned, including but not limited to negativeselection, continuous, and series operation, all of which will bediscussed in greater detail below.

The MSFB

The MSFB used in this method is comprised of a column which is packedwith magnetizable beads or other magnetizable particles, means forfluidizing the bed, and means for subjecting the bed to a magneticfield. The method of use is as described by the manufacturers withsuitable modifications as described herein. A typical MSFB is shown inFIG. 1. As shown in FIG. 1, magnetizable beads or particles (12) areplaced in a tubular column (14). A distributor plate or screen (16)having a plurality of apertures (18) which are smaller than themagnetizable particles is used for retaining the particles in the columnand for distributing a fluid upwardly through the column. The bottom ofthe column is fitted with at least one piping connection (20) forplacing a fluidizing medium in fluid communication with the column.Another connection (22) may be provided for removing the particles fromthe bottom of the bed.

Similarly, the top of the column is also fitted with at least one pipingconnection (24). This connection may be used for removing the fluidizingmedium after it has passed through the bed. Alternatively, thisconnection could be used for washing the bed to remove cells which donot bind to the particles or to remove cells that have been releasedfrom the particles. Furthermore, this connection could be used forremoving the particles,. either with or without bound cells. Anotherpiping connection (26) may be provided at the top of the column for theaddition of particles to the top of the bed during continuous modeoperation.

The size of the MSFB column should be designed to accommodate the enduse of the mammalian cells being separated in the MSFB. For example, ifthe MSFB is being used to separate mammalian stem cells for clinicalpurposes, then the size of the MSFB is likely to be in the range of 30to 500 ml. For separation of cells for use in laboratory experiments,the size of the MSFB can be significantly smaller, typically in therange of 1 to 10 ml.

Means for forcing a fluid upwardly through the column are required forthe operation of the MSFB. This may include at least one pump (28), atleast one valve (30), (31), and (32), and piping (34) for connectingvarious fluids to the MSFB. As an alternative to a pump, a gravity-fedor pressurized tank may be used to drive the fluid through the MSFB. Thefluids or solutions which may be passed through the MSFB may include thecell suspension containing the mixture of cell populations of mammaliancells (36), a wash solution (38), elution buffers (40), or othersolutions such as a solution which is used to initially fluidize the bedbefore the cell suspension is passed through the bed. Such solutions areknown in the art and should be isotonic, preferably sterile andpreferably buffered to remain within a nontoxic pH range.

Means for subjecting the column of particles to a magnetic field is alsorequired for the operation of the MSFB. A substantially uniform magneticfield may be created by any suitable means including, but not limitedto, at least two coils which surround the column. As shown in FIG. 1,one embodiment includes a modified Helmholtz coil having an upper coil(42) and a lower coil (44), both which surround the column. A powersupply (46) is connected to both coils. Alternatively, a permanentmagnet or any device which is capable of generating a substantiallyuniform magnetic field can be used in this invention.

The magnetic field should be substantially uniform in both the radialand axial directions along the length of the column. While the use of asubstantially uniform magnetic field is preferred, this invention alsocontemplates the use of non-uniform magnetic fields.

The strength of the magnetic field will depend on severalcharacteristics of the magnetizable particles used, including the typeand amount of magnetic material used and the size and density of theparticles. A magnetic field in the range of 10 to 200 gauss is typicallyrequired to stabilize a fluidized bed. A determination of the suitablemagnetic field strength is within the skill of one in the art.

The magnetizable particles for use in the MSFE can be made frommagnetic, paramagnetic or superparamagnetic materials. Alternatively,they can be a composite of a magnetizable material and a non-magneticmaterial. Suitable magnetizable material includes, but is not limitedto, magnetite, and nickel, iron, copper, cobalt and their alloys.Magnetite is the preferred material. Non-magnetic materials that aresuitable for use in the composite particles include any of the solidsupport materials. These include, but are not limited to, sepharose,sephadex, agarose, polymers such as polystyrene and acrylic, and anyother biocompatible material. For a composite particle, the magnetizablematerial can be mixed with the support material and formed intoparticles. Alternatively, a core of magnetizable material can be coatedwith a uniform layer of the non-magnetic material. In general, the outercoating should be a relatively uniform layer that completely covers andhides any irregularities in the magnetic core. The thickness of thecoating will typically be in the range of about 0.1 microns to 100microns. The choice of the non-magnetic material selected for the outercoating is dependant on the type of affinity binding pair selected forthe particular separation. Once a binding pair is chosen for theparticular target cell to be separated, the determination of thesuitable support material for the outer coating is within the skill ofone in the art. In general, the magnetic material should compriseapproximately 2 to 98% by volume of the total volume of the compositeparticle.

In yet another embodiment of the invention, magnetizable particles ormagnetizable/non-magnetic composite particles can be used in conjunctionwith separate non-magnetic particles. In this alternative configuration,the magnetizable particles should comprise approximately 35 to 70% byvolume of the total volume of particles in the column. See Chetty,"Overcoming Support Limitations in Magnetically Stabilized Fluidized BedSeparators", Powder Technology, 64:165-174 (1991).

The magnetizable particles must be designed to allow a fluidizationvelocity which will allow for binding of a specific mammalian cellpopulation to the particles. In general, the fluidization velocity is afunction of the diameter and density of the particle and the fluiddensity and viscosity. The fluidization velocity of particles must becalculated to insure that the flow rate required to fluidize theparticles is not too high. Too high a flow rate will result in lessbinding and increased filtration of cells. A determination of thesuitable fluidization velocity is within the skill of one in the art.

Another factor which must be considered when determining the appropriateparticle size is the size of the cell to be bound to the particle. Inthis method, mammalian cells having a size in the range of approximately8 to 20 microns will desirably bind to the particle. One problem whichmay occur is that, if small particles are used, once the selected cellbinds to the particle, the density of the combined cell/particle complexmay be increased to a point where the cell/particle complex flows out ofthe column. One solution to this problem is, to increase the magneticfield strength to hold the cell/particle complex in place.Alternatively, the cell/particle complex can be allowed to pass throughthe column and can be separated outside the column using a simple magnetor magnetic field.

Preferably, the diameter of the magnetizable particle is much greaterthan the diameter of the cell in order to increase the bindingprobability and capacity. In view of these factors, the inventors havefound that magnetizable particles having a size in the range of 5 to 500microns are suitable for mammalian hematopoietic cell separations.Preferably, the particles have a size in the range of about 150 to 350microns; more preferably, the particles have a size of about 250±50microns.

The density of the magnetizable particles should be in the range ofabout 1 to 10 g/cm³. Preferably, the particles have a density in therange of about 1.5 to 5 g/cm³.

Operation of the MSFB

In one embodiment of this invention, the MSFB is operated by firstfluidizing the bed with a solution. Fluidization occurs when, at aspecific flowrate, the fluid drag on the particles overcomes gravity andthe particles lift off the bottom of the column. The solution used toinitially fluidize the bed may be a wash solution or any other fluid.Once the bed is fluidized, the bed is subjected to a radially andaxially substantially uniform magnetic field. The effect of the magneticfield can be thought of as creating a magnetic dipole in each particle,which causes each particle to remain fixed in place in a directionparallel to the magnetic field lines. Finally, once the bed is fluidizedand magnetically stabilized, the suspension containing the mammaliancells is passed through the bed for separation. Those skilled in the artwill recognize that it may be desirable to fluidize the bed using thecell suspension instead of using a different solution. Alternatively, itmay be desirable to first magnetically stabilize the bed beforefluidization.

As described above, the velocity at which the suspension is passedthrough the bed is limited by the ability of a specific mammalian cellto be sufficiently in contact with the particle so as to bind to thatparticle. In addition, the velocity must be low enough so that cellsbound to the particles are not removed from the particles by fluid drag(shear forces). The fluidization of the bed allows for a constantvelocity of the suspension through the bed, which also assists in theindividual cells binding to the support particles.

The fluidization velocity must also be designed to achieve an optimumexpansion of the bed. Typically, an optimum bed expansion is in therange of about 2 to 50% of the original bed volume. The optimum size ofthe expanded bed is dependent on the number of target cells sought to beseparated. For example, typically about 109 CD34⁺ cells can be obtainedfrom a normal apheresed peripheral blood sample (typically about 50 to500 ml, to which an equal volume or more of media is added). In order tobe able to positively select this number of cells, the total volume ofthe expanded bed should be in the range of about 50 to 500 ml.

In general, if the viability of the cells is a concern, the MSFB shouldbe operated under conditions that support cell life. Typically, thiswill require an operating temperature in the range of about 4° C. toabout room temperature (27°-28° C.). Additionally, while the MSFE willnecessarily be under some pressure do to the fluidization of the bed,this pressure should be kept to a minimum, and only as high as necessaryto operate the MSFB.

When the suspension containing the mixture of cells is depleted orotherwise stopped, a wash solution may be passed through the column, ifdesired, to wash either bound or unbound cells from the bed. The washsolution may be introduced either through the top or the bottom of theMSFB column. If a positive selection method using affinity binding wasperformed, the bound target cell population can be released by eitherphysical or chemical methods as discussed below. If a positive selectionmethod was performed using the magnetic attraction between cellscomplexed with magnetizable superparamagnetic nanoparticles and themagnetizable particles, then the cells can be released from themagnetizable particles by simply removing the magnetic field and washingthe now unbound cells from the MSFB column. Similarly, if a negativeselection method was performed, the bound nontarget cells could also bereleased through either physical or chemical methods, or by removing themagnetic field, if they are desired for some use.

If the viability of the cells is a concern, the bound cells must beseparated from the particles using chemical methods that do not harm thecells. Examples of chemical methods include, but are not limited to,agents that reduce, oxidize, or otherwise chemically alter a chemicalspecies, agents that effect a change in pH, or agents that changeosmolality.

Blood cells are exquisitely sensitive to changes in pH, which must becarefully controlled to maintain cellular integrity. The normal pH ofblood is 7.35-7.45. Nevertheless, a change in pH of the solutions in thefluidized bed may affect the affinity chemistry and decrease cellularadherence without killing the cells. Such a change in pH can be effectedby adjusting the pH of a second elution buffer to be passed through theMSFB. In one example, the pH of the blood cell suspension is at about7.25 and the pH of the second solution is at about 7.00. In anotherexample, the pH of the blood cell suspension is at about 7.25 and the pHof the second solution is at about 6.80. In another example, the bloodcell suspension has a normal pH, and the pH of the second solution issufficiently low or high to permit release of the cell.

In a different example, the blood cell suspension has an elevated ordepressed pH (for example, greater than 7.45 and less than 7.35,respectively), and the second solution has a normal pH. These values canbe adjusted by routine experiments by those skilled in the art, tomaintain cell viability and increase selectivity. After the bound cellsare released, the pH of the cell suspension is adjusted to normal by theaddition of the appropriate dilute acid or base. In addition, the cellsuspension may be buffered using any suitable physiologically acceptablebuffers including, but not limited to, any dibasic or monobasicphosphate saline, sodium bicarbonate, phosphate buffered saline (PBS),and (N- 2 Hydroxy-ethyl!piperazine-N'- 2-ethanesulfonic acid! (HEPES).

Chemical separation may also include competitive binding techniques orchemical alteration of the substance which binds the mammalian cell tothe particle.

Physical separation methods may include decreasing or otherwise stoppingthe magnetic field to allow radial and axial movement of the particlesin the bed. The resulting Brownian motion will result in release of thebound cells from the particles. Alternatively, the velocity of a secondsolution, most probably a wash solution or elution buffer may beincreased significantly to permit large enough hydrodynamic shear forcesto separate the cell from the particles. Furthermore, a combination ofdecreasing the magnetic field and passing a second solution through thebed at an increased velocity may be used to allow the combination ofrandom motion and shear forces to separate the bound cells from theparticles.

This MSFB system, as described above, could have significant advantagesin positive selection. This method does not result in significantnon-specific cell separation because the contaminants are not trapped bythe bed of particles. Because of the fluidization of the is bed ofparticles, only those mammalian cells having affinity for the bindingsubstance attached to the particle will bind to the particle. Theremaining cells will pass through the bed and can be collected fordisposal, additional separations or other processing, as desired. Thismethod also allows for very high efficiency capture of a positivelyselected population with minimal losses. This significantly decreasesthe necessity for downstream purification, if desired, due to theincreased purity of the sample.

Modes of Operation:

Several possible modes of operation of a MSFB with specific applicationto BM or PB are contemplated. These include:

(a) Target cell populations, for example CD34⁺ cells, could bepositively selected by binding to particles in the MSFB while thenon-target cells pass through the MSFB and are discarded. The particlescould be washed by flowing a wash buffer through the fluidized,magnetically stabilized bed, if required. The selected target cellpopulation could then be eluted from the fluidized, magneticallystabilized bed using the appropriate elution buffer. The collected cellscould then be recovered from the elution buffer.

(b) A negative selection mode could be used to bind nontarget cellpopulations to the magnetizable particles in the MSFB. Examples of cellswhich are suitable for a negative selection method include, but are notlimited to, the mature cell lineage panel having markers including, butnot limited to, CD2, CD14, CD15, CD19. An example of lineage specificmarkers is presented in Table 1. These mature nontarget cells would bindto the particles while the target cell population passes through to becollected. The particles in the MSFB could then be washed with bufferand the additional target cell population collected.

Table 1 summarizes probable phenotypes of stem cells in fetal, adult,and mobilized peripheral blood. In Table 1 myelomonocytic stands formyelomonocytic associated markers, NK stands for natural killer cellsand AMPB stands for adult mobilized peripheral blood. As used hereinboth infra, supra and in Table 1, the negative sign or, uppercasenegative sign, (⁻) means that the level of the specified marker isundetectable above Ig isotype controls by FACS analysis, and includescells with very low expression of the specified marker.

                                      TABLE 1                                     __________________________________________________________________________    Probable Stem Cell Phenotypes                                                 NK           B cell            Other                                          and T cell markers                                                                         markers  Myelomonocytic    HLA-        P-gp                      CD2    CD3                                                                              CD8                                                                              CD10                                                                             CD19                                                                             CD20                                                                             CD14                                                                             CD15                                                                             CD16                                                                             CD33                                                                             CD34                                                                             CD38                                                                             DR C-Kit                                                                            Thy                                                                              Rho                                                                              Activity                  __________________________________________________________________________    FBM -  -  -  -  -  -  -  -  -  ?  +  -  +  +  +  lo +                         ABM -  -  -  -  -  -  -  -  -  -  +  ?  lo/-                                                                             +  +  lo +                         AMPB                                                                              -  -  -  -  -  -  -  -  -  lo/-?                                                                            +  ?  lo/-                                                                             ?  +  lo +                         __________________________________________________________________________

(c) The MSFB could be run in batch mode, or in continuous mode bycontinually supplying particles into the top (or other point) of thecolumn and removing them from the column bottom (or other point). Thiswould allow use of a small column to process a whole BM aspirate or PBapheresis.

(d) For a negative selection, binding substances having affinity for themature cell lineage panel could be attached to the particles in a singleMSFB. Alternatively, a plurality of binding substances each havingaffinity for a different mature cell could be used individually in aseries of MSFBs with the cell suspension flowing sequentially throughthe various MSFBs.

(e) "Prepackaged" sets of MSFB columns could be linked together inseries or parallel for any combination of selections. For example, aseries of MSFB columns could be used in a negative selection mode tosequentially remove a series of nontarget cells. Alternatively, a set ofMSFB columns could be used in either series or parallel to positivelyselect a group of target cell populations by binding these differenttarget cell populations to particles in the various prepackaged MSFBcolumns. Finally, a set of MSFB columns could be used in series orparallel to both positively and negatively select any number of cells.These various combinations of prepackaged MSFB columns will allow forvery simple patient-specific cell selection/removal.

(f) Alternatively, for batch mode separation, the MSFB could be filledwith particles and cells and slowly rotated to a horizontal position.The magnetic field is not activated during the actual selection step, inwhich the cells contact and adhere to the particles for capture. TheMSFB could then be rotated to a vertical position and the magnetic fieldactivated to recover the cells in a typical MGFB mode. This is depictedin FIG. 2.

(g) The MSFB could be used to select and separate more than one type ofmammalian cell. In this configuration, a portion of the MSFB could befilled with magnetizable particles having affinity for one type of cell,while another portion is filled with magnetizable particles havingaffinity for a different type of cell. While these two types ofmagnetizable particles could be dispersed throughout the column, it maybe desirable to physically separate the portions between the top and thebottom sections of the column. This latter configuration can beaccomplished by using magnetizable particles of different densities orsize. For example, one portion of the particles could have a density of2 g/cm³ while a second portion could have a density of 4 g/cm³. Thesetwo types of particles will segregate in the MSFB during fluidizationbefore the magnetic field in applied. In another example, particles thathave the same density but which have different diameters, such as 300microns and 150 microns, could be employed.

In this mode of operation when using either particles of differentdensities or size, the MSFB effectively has two separate beds in asingle column. In alternate configurations, the MSFB could have morethan two portions, each having magnetizable particles of differentdensity or size.

This system has the potential to significantly improve the debulking ofBM or PB harvests by either the positive selection of a target cellpopulation or the negative selection of cells other than the desiredtarget cell population. In addition, because this method can be used forpositive selection of a target cell population, the time and reagentsneeded for later processing, such as staining and sorting of the cells,can be drastically reduced.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the claimsset forth below.

We claim:
 1. A method for the selective enrichment of at least onemammalian target cell population from a suspension of a mixture of cellpopulations wherein said suspension contains said target cell populationand at least one nontarget cell population, said method comprising thesteps of:attaching a plurality of magnetizable nanoparticles havingaffinity for at least one specific population of mammalian cells to saidspecific populations of cells; fluidizing a column containing a bed ofmagnetizable particles with a first solution; stabilizing said fluidizedbed of magnetizable particles with a magnetic field; and passing thesuspension through said fluidized, magnetically stabilized bed at avelocity at which at least one specific population of cells binds tosaid magnetizable particles through magnetic attraction between saidnanoparticles and magnetizable particles, thereby enriching the targetcell population.
 2. The method according to claims 1 wherein saidmagnetizable nanoparticles have affinity for said target cell populationand the method further comprises the step of collecting said target cellpopulation bound to said particles.
 3. The method according to claim 2wherein said collecting step further comprises the steps of releasingsaid target cell population by removing the magnetic field and washingthe unbound target cell population from said bed.
 4. The methodaccording to claim 1 wherein said magnetizable nanoparticles haveaffinity for said nontarget cell population and the method furthercomprises the step of collecting said target cells which pass throughsaid fluidized, magnetically stabilized bed.